The proposed research is focused on the development of high frequency dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) to enhance sensitivity in NMR based structural biology experiments. In addition, we plan time domain EPR spectroscopy to provide support for the DNP research. 1 Dynamic Nuclear Polarization 1.1 CW DNP at 140, 250, 330 and 460 GHz During the coming grant period our efforts will focus on extending the DNP to 330 GHz/500 MHz and 460 GHz/700 MHz which are frequencies used in contemporary protein NMR. This effort will include developing: (1) low temperature, quadruple resonance MAS probes, (2) helium cooling for low temperature MAS experiments, and (3) implementing tunable gyrotrons. 1.2 Applications to Peptides and Proteins We plan to continue application of DNP to studies of proteins with the goal of optimizing the signal enhancements for 2H labeled proteins where we have recently observed 5=120-140, which is ~x4 larger than for 1H species. Experiments are planned to determine the optimal polarizing agent and irradiation field for 2H,13C,15N labeled nanocrystalline and amyloid proteins. We will also investigate the structure of H2O in amyloid fibrils with 17O DNP experiments. 1.3 Polarizing Agents We plan to continue to develop new polarizing agents with studies of: (1) a H2O soluble bTbk;(2) trityl- TEMPO (3) BDPA-TEMPO and (4) Trityl-BDPA mixtures and biradicals. 1.4 Time Domain DNP Experiments: Pulsed DNP mechanisms do not display the B0 dependence seen in CW experiments. For this reason we will be investigating the integrated solid effect (ISE), the dressed state solid effect (DSSE), and NRF- DNP and NOVEL. We plan to perform these experiments at 9 GHz, where it is easy to control microwave pulses and phases, and at 140 GHz using our renovated spectrometer. 1.5 TJ-DNP Experiments: We plan to refine the TJ-DNP by using a 1.5 5m laser and sapphire rotors that should accomplish melting more rapidly. The experiments are also applicable to heating proteins in MAS spectra to temperatures where the resolution is improved. 2 High Frequency EPR : 9 and 140 GHz EPR spectra will be used to characterize the paramagnetic centers used for DNP and understand the DNP mechanisms. PUBLIC HEALTH RELEVANCE: The research proposes to use dynamic nuclear polarization to improve the sensitivity in magic angle spinning NMR experiments. The higher sensitivity will lead to molecular structures with higher precision and could have a profound impact on structural biology, solution NMR and medical imaging. In addition, we propose to develop new polarizing agents for DNP, new equipment for the experiments, and new time domain methods for more rapid data acquisition. The experiments will be applied to a variety of model and unknown systems whose structure we will determine.